1. Field of the Invention
The present invention is related to a human tyrosine hydroxylase (TH) promoter and uses thereof.
2. Background of the Invention
A number treatment methods are currently under investigation where healthy cells performing a specialized function are transferred, either directly or after genetic modification, to an individual to treat a disease condition. Such diseases include neurological diseases, leukemia, and immune and genetic disorders. Specifically, cell based therapies have been described or suggested for type I insulin dependent diabetes (Bonner-Weir et al. J Pathol 2002; August 197(4):519–26); adenosine deaminase (ADA)-deficient severe combined immunodeficiency (SCID) (Aiuti et al. Science 2002; Jun. 28, 296(5577):2410–3); Parkinson's disease (Arenas, Brain Res Bull. 2002 April; 57(6):795–808); and mucopolysaccharidoses, a group of lysosomal storage diseases (Vogler et al., Pediatr Dev Pathol 2001 September–October; 4(5):421–33). Cell replacement therapy has also been contemplated for treatment of other diseases such as stroke, head and spinal cord trauma, Alzheimer's Disease, multiple sclerosis, amyotrophic lateral sclerosis (ALS), genetic enzyme deficiencies in general, and muscular dystrophy (see, e.g. Holden, Science, 297:500–502, 2002).
However, the source of such replacement cells and purification procedures to enrich such cells from the sources are not always optimal.
For example, in Parkinson's disease, the degeneration of the TH+ dopaminergic cells of the substantia nigra parallels the symptoms. Cell replacement or transplantation has been hindered by the limited supply of cells as up to 6–8 fetal brains are harvested for treatment of a single patient. Furthermore, the TH+ dopaminergic cells represent only 0.5–1% of the fetal mesencephalon. This may well underlie the variability and low success rate of this treatment. Therefore, for the purposes of cell-transplantation treatments, it would be desirable to develop methods to easily identify, isolate and enrich the population of TH+ dopaminergic cells.
Similar to neurons, the endocrine cells of the mammalian pancreas have been considered to be post-mitotic, i.e., terminal, essentially non-dividing cells. Recent work has shown that the cells of the mammalian pancreas are capable of survival in culture, however, propagation of differentiated cells having endocrine function has met with, at best, limited success. In addition, pancreatic ductal cells have been differentiated into islet cells (Ramiya et al., 2000) and also human embryonic stem cells have been shown to be capable of differentiating into pancreatic islet cells (Lumensky et al. 2001).
However, problems in isolation of the proper insulin-producing population from the cell cultures limits their use in treatment of pancreatic disorders. Such disorders include diabetes mellitus, a disease that impairs or destroys the ability of the beta cells of the islets of Langerhans (structures within the pancreas) to produce sufficient quantities of the hormone insulin, a hormone that serves to prevent accumulation of sugar in the bloodstream. Type I diabetes mellitus (insulin dependent, or juvenile-onset diabetes) typically requires full hormone replacement therapy. In type II diabetes, sometimes referred to as late onset, or senile diabetes, treatment often does not require insulin injections because a patient suffering with Type II diabetes may be able to control his/her blood sugar levels by carefully controlling food intake. However, as many as 30% of these patients also have reduced beta cell function and, therefore, are candidates for hormone replacement therapy as well.
Current treatment of individuals with clinical manifestation of diabetes attempts to emulate the role of the pancreatic beta cells in a non-diabetic individual. Individuals with normal beta cell function have tight regulation of the amount of insulin secreted into their bloodstream. This regulation is due to a feed-back mechanism that resides in the beta cells that ordinarily prevents surges of blood sugar outside of the normal limits. Unless blood sugar is controlled properly, dangerous, even fatal, blood sugar levels can result. Hence, treatment of a diabetic individual nowadays involves the use of recombinantly produced human insulin on a daily basis.
Injected insulin and diet regulation permit survival and in many cases a good quality of life for years after onset of the disease. However, there is often a gradual decline in the health of diabetics that has been attributed to damage to the vascular system due to the inevitable surges (both high and low) in the concentration of glucose in the blood of diabetic patients. In short, diabetics treated with injected insulin cannot adjust their intake of carbohydrates and injection of insulin with sufficient precision of quantity and timing to prevent temporary surges of glucose outside of normal limits. These surges are believed to result in various vascular disorders that impair normal sight, kidney, and even ambulatory functions.
Both of these disease states, i.e., type I and type II diabetes, involving millions of people in the United States alone, preferably should be treated in a more regulated fashion. Successful transplants of whole isolated islets, for example, have been made in animals and in humans. However, long term resolution of diabetic symptoms has not yet been achieved by this method because of a lack of persistent functioning of the grafted islets in situ (see Robertson (1992) New England J. Med., 327:1861–1863).
For the grafts accomplished thus far in humans, one or two donated pancreases per patient treated are required. Unfortunately only some 6000 donated human pancreases become available in the United States in a year, and many of these are needed for whole pancreas organ transplants. Therefore, of the millions of diabetic individuals who could benefit from such grafts, only a few of them may be treated given the current state of technology. If the supply of insulin producing islet cells could be increased by culturing and/or differentiating pluripotent stem cells they would provide much needed material to allow development of new cell replacement treatment options for diabetes. Therefore, it would be useful to develop a method of culturing cells that could be differentiated into pancreatic islet cells and a method that would allow easy selection of insulin-producing cells from the cultures for the purposes of administering such cells to individuals in need thereof.
The enzyme tyrosine hydroxylase (EC 1.14.16.2, TH) is encoded by a single copy gene on human chromosome 11p158; 51 and it catalyzes the rate limiting step in the synthesis of catecholamine neurotransmitters in the central and peripheral nervous systems: the hydroxylation of tyrosine to yield dopa.45 For example, islet cells have been shown to contain TH enzymes involved in the synthesis of catecholamines (Teitelman, et al., Development 1993, 118: 1031–1039; Iturriza, et al., Neuroendocrinology 1993, 57: 476–480). Thus far, regulation of TH expression has been studied mainly because of its role in biosynthesis of catecholamines in neurophysiology and the alterations associated with a variety of psychiatric illnesses. Classically, drugs effective for treatment of psychotic symptoms have been antagonists of dopamine receptors, and a number of studies have assessed the linkage between mutations in the TH gene and disorders such as schizophrenia and bipolar disorder. However, there is no evidence as of yet for a role in these, or other diseases of mutations in the TH protein coding regions. However, polymorphisms elsewhere in the TH sequence, and in TH-regulating transcription factors have been described.38, 5 
In light of the above, it would therefore be advantageous to discover the functional domains regulating human TH-gene expression, particularly regulatory regions directing human TH expression to both central and peripheral neurons.